| Peritoneal Dialysis(PD)is one of the main methods of renal replacement therapy,.However,Peritoneal fibrosis(PF)is an important reason for patients to withdraw from Peritoneal dialysis,in which the abdominal differentiation of membrane epithelial cells into mesenchymal cells(EMT)is the starting link of Peritoneal fibrosis.Previous studies have shown that indoxyl sulfate(IS)can be lured the occurrence of EMT in Peritoneal mesothelial cells,but whether β-catenin involvement has not been clear,this topic mainly to is caused by the EMT process further in-depth study,whether molecules participate in peritoneal skin cells that occur EMT,and use β-catenin small molecular inhibitors(ICG-001)intervene to see if it can reverse the occurrence of EMT,aiming for Peritonealfibrosis provides new methods.[Objective]1.To Culture human Peritoneal mesothelial cells immortalized cell line(HMrSV5)in vitro,Using IS stimulates HMrSV5 cells,using 4.25 % glucose Peritoneal dialysis fluid(PDF)as a Positive control,observering cell morphology by microscope,detecting the expression of E-cadherin,β-catenin and α-SMA;2.Add β-catenin small molecular inhibitors(ICG-001)to the cell groups of the IS and PDF,to detect the expression of the above molecules,observe its effect on the EMT of Peritoneal skin cells,and explore whether the β-catenin molecule is involved in the Process of EMT occurrence,ICG-001 can reverse EMT;3.Using cellular function experiments: cell matrix adhesion experiment and CCK-8 analysis experiment to detect different the adhesion and Proliferative ability of group cells to further verify whether IS causes Peritoneal skin cells to occur EMT;[Methods]1.Human Peritoneal mesothelial cell line(HMrSV5)was cultured in vitro and resuscitated and passaged.The cells were divided into 5 groups:(1)Control group(RPMI1640+10%FBS medium,no intervention during the culture);(2)IS group(IS culture with a concentration of 1000umol/L and RPMI1640+10%FBS medium);(3)containing 5umol/L ICG-001+1000umol/L IS co-cultured with RPMI1640+10%FBS medium;(4)PDF group(4.25% Peritoneal fluid co-cultured with RPMI1640+10%FBS medium);(5)containing 5umol/L ICG-001+4.25% PD fluid with RPMI1640+10%FBS medium;2.The cells were cultured at 0hours,12 hours,24hours and 48 hours,72 hours,the morphological changes of cells in each group were observed,and through the methoeds of real-time PCR and western blot to detect the exPression of the gene and Protein of E-cadherin、β-catenin、α-SMA;3.Cellular function experiments: cell matrix adhesion experiment and CCK-8 analysis experiment to detect different the adhesion and Proliferative ability of group cells;[Results]1.Compared with the normal group,stimulated by the IS,cells become thinning and lengthening,and the change of the spindle shape,and the cells of β-catenin inhibitor(ICG-001)intervention,the morphological changes of cells are not obvious;2.β-catenin and α-SMA is hardly expressed in the cells of the control group(Culture Liquid Group),after IS,PDF stimulates cell 24 h later,the expression of β-catenin and α-SMA significantly increased;and in the control group,the expression of E-cadherin in the IS group and the PDF group decreased significantly from 24 h to decrease,and the difference showed statistically significant(P<0.05);3.Compared with the IS group and the PDF group,the β-catenin inhibitor group in 24 h,48h,72 h,the expression of E-cadherin is ascended,the expression of β-catenin and α-SMA is decreased,it is statistically significant(P<0.05);4.The results of cell matrix adhesion experiment showed that IS and PDF group adhesion cells higher compared with the normal control group.There was a significant increase in the number of adhesion cells observed in the IS/PDF+ICG-001 group,and the difference was statistically significant(P< 0.0001);5.The results of the CCK-8 experiment showed that in 72 h,IS and the PDF group and the normal control group compared the value of measured under 450 nm absorption light increased,while IS/PDF+ICG-001 group detected the OD value decreased compared with the former,which was statistically significant(P<0,0001);[Conclusion]1.Using IS to stimulate peritoneal mesothelial cells,Cell function experiments show that IS stimulated by peritoneal mesothelial cells,cells-Matrix adhesion capability showed increased and the proliferative capacity of cells increased accordingly,can make cell morphology change;at the same time can promote α-SMA gene and protein expression increased,E-cadherin gene and protein decreased The reduction indicates that the IS can induce EMT in the peritoneal cells;2.IS induced peritoneal cells to occur in the EMT process,β-catenin’s expression rises,after the use of β-catenin inhibitors,β-catenin,α-SMA expression drops,Ecadherin’s expression rises,reduce the cells-Matrix adhesion capability and Proliferative activity,prompts β-catenin to participate in IS induced by peritoneal mesothelial cells to have EMT process;... |
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